PROJECT SUMMARY In our continued efforts to understand the biology of Influenza A virus (IAV), we have characterized RNA from both the host and virus using next generation sequencing. These efforts led to the discovery of a small virus- derived RNA (svRNA) that was necessary for mediating the viral switch from transcription to replication. Upon characterizing the biology of svRNA, we documented the surprising finding that svRNA synthesis was dependent on the expression of NEP, a minor protein of the virus that slowly accumulates in the cell as a result of inefficient splicing. Together, these two discoveries suggested NEP may be the master regulator of IAV infection. In short, we found that the slow accumulation of NEP provided the virus with a ?timer? to coordinate svRNA production and the subsequent switch from transcription to replication. Furthermore, we, and others, have found that NEP-mediated svRNA synthesis is dependent on its capacity to induce cRNA. Despite determining this function for NEP, how it coordinates this activity remains entirely unknown. Here we seek to understand this activity through three complementary aims. Aim 1 details a strategy to ascertain how NEP interaction with the RNA dependent RNA polymerase influences its activity. Aim 2 seeks to ascertain why NEP is associating with host factors involved in RNA processing. Aim 3 investigates the impact of NEP and its associated proteins on virus pathogenicity and tropism. The experimental strategy comprising these aims will reveal exciting new molecular targets that can be exploited to generate a novel class of antivirals and will significantly increase our understanding of IAV replication.